Vitamin D is an essential fat-soluble vitamin group mainly for higher organisms and formed by biosynthesis from cholesterol. Vitamin D plays important roles in organisms due to its wide variety of physiological activities such as stimulating absorption of calcium, metabolic stimulation, inducing cellar differentiation and immune regulation and the like.
In a main vitamin D biosynthetic pathway in human beings, after 7-dehydrocholesterol (provitamin D3) is first synthesized from cholesterol, vitamin D3 is produced in the skin by ultraviolet ray and thermal reaction. 25-hydroxy vitamin D3 is obtained by 25-hydroxylation of vitamin D3 by mitochondrially-located cytochrome P450 (CYP27A1) in the liver. Then, through further 1α hydroxylation by another cytochrome P450 (CYP27B1) in the proximal convoluted tubule, 1α,25-dihydroxy vitamin D3 (activated vitamin D3) is produced. It is confirmed that this substance regulates expression of specific nuclear genes involved in expression of physiological activities by being bound to an intracellular receptor.
Accordingly, when the liver or kidney becomes dysfunctional, normal vitamin D metabolism may be inhibited. Regarding the patient with such symptoms, the level of 25-hydroxyvitamin D3 or 1α,25-dihydroxy vitamin D3 in the blood may extremely reduces, and it is necessary for treatment to supplement 25-hydroxyvitamin D3 or 1α,25-dihydroxy vitamin D3 by the administration.
Meanwhile, rickets is known as being caused by a low level of 1α,25-dihydroxy vitamin D3 due to dysfunction of hydroxylation resulting from a congenial genetic mutation in cytochromes which is involved in hydroxylation of vitamin D3. 1α,25-dihydroxy vitamin D3 and analogous compounds having similar physiological activities have significant importance as therapeutic agents for diseases due to lack of 1α,25-dihydroxy vitamin D3 including acquired rickets and osteoporosis as well as congenial rickets.
Furthermore, owing to various physiological activities of the vitamin D group, various derivatives thereof have been studied as candidate agents and developments of antineoplastic agent, antipsoriatic agent, immunostimulating agent and the like are also expected. Seen from this view point, hydroxylation is important as one method for modifying by derivatizing the vitamin D group. In such a case, the importance of hydroxylation is not limited to 1α- and 25-positions but great needs may arise for hydroxylation at other positions. To meet such needs for manufacturing and discovering drugs, there has been rising demand in the pharmaceutical industry for improving production method of a hydroxide of vitamin D and the like in order to manufacture and supply a hydroxide of vitamin D more efficiently at lower cost.
Referring to 1α,25-dihydroxy vitamin D3, which has particularly strong physiological activities and therefore has high value among the vitamin D group, the production method thereof includes an organic synthesis method, a method using cytochromes P450 of human beings and a method using hydroxylase of microorganisms. Among these, a method for synthesizing 1α,25-dihydroxy vitamin D3 from cholesterol through 17 steps has been known in the organic synthesis method. In the method using cytochromes P450 of human beings, biological and biochemical findings have been accumulated relating to genes involved, technology for expression and the nature of cytochromes P450 enzyme (Review by T. Sakaki et al., Frontiers in Bioscience, 10, 119-134, 2005; Non-patent document 1). However, both of the two methods are quite unsuitable for practical use owing to high production costs and low productivity.
Meanwhile, a method using hydroxylase of microorganisms is a relatively promising production method.
For example, Sasaki et al. of Taisho Pharmaceutical Co., Ltd. isolated actinomycete, Pseudonocardia autotrophica, producing 1α,25-dihydroxy vitamin D3 by hydroxylation of vitamin D3 in the course of search for microorganisms (J. Sasaki et al., Applied Microbiology and Biotechnology, 38, 152-157, 1992; Non-patent document 2). As a result of improvement in breeding of the strain and development of the production processes at Mercian Corporation, the method for producing 1α,25-dihydroxy vitamin D3 by microbial conversion was established (K. Takeda et al., J. Ferment. Bioeng., 78, 380-382, 1994: Non-patent document 3) and has been put to practical use.
Laid-Open Japanese Patent Publication No. 2003-325175 (Patent Document 1) by Mitsubishi Chemical Corporation discloses a method for producing 25-hydroxy vitamin D3 by hydroxylation at 25-position of vitamin D3 using Bacillus megaterium. However, the amount of accumulated 25-hydroxy vitamin D3 in the culture medium is small by this method and the publication does not describe the production of 1α,25-dihydroxy vitamin D3.
Regarding the finding on a gene for the vitamin D3 hydroxylase derived from microorganisms, a gene for vitamin D3 hydroxylase at 25-position derived from Pseudonocardia autotrophica is reported in 1994 (H. Kawauchi et al., Biochimica et Biophysica Acta, 179-183, 1994; Non-patent document 4), which is apparently different from the gene of the present invention in terms of the nucleotide sequence and the characterization of the enzyme encoded by the gene. Also, Sawada et al. reported in 2004 that P450SU-1 (CYP105A1) encoded by cytochromes P450 gene derived from actinomycete, Streptomyces griseolus ATCC 11796, showed weak activity for hydroxylation of vitamin D3 to 25-hydroxy vitamin D3 and that of 25-hydroxy vitamin D3 to 1α,25-hydroxy vitamin D3 D. P. O'Keefe et al., Arch. Microbiol., 149, 406-412, 1988; Non-patent document 5).
Thus, various findings have been shown on microbial transformation of vitamin D3 by hydroxylation. Among these, only the method for producing 1α,25-dihydroxy vitamin D3 from vitamin D3 using Pseudonocardia autotrophica strain for transformation has been put into practical use industrially (Laid-open Japanese patent publication H02-469 (U.S. Pat. No. 4,892,821); Patent document 2 and Laid-open Japanese patent publication H02-231089; Patent document 3). However, issues to be solved by improvement for a more efficient production process still remain in this method.    [Non-patent document 1] Review by T. Sakaki et al., Frontiers in Bioscience, 10, 119-134, 2005    [Non-patent document 2] J. Sasaki et al., Applied Microbiology and Biotechnology, 38, 152-157, 1992    [Non-patent document 3] K. Takeda et al., J. Ferment. Bioeng., 78, 380-382, 1994    [Non-patent document 4] H. Kawauchi et al., Biochimica et Biophysica Acta, 179-183, 1994    [Non-patent document 5] D. P. O'Keefe et al., Arch. Microbiol., 149, 406-412, 1988    [Patent document 1] Laid-Open Japanese Patent Publication No. 2003-325175    [Patent document 2] Laid-open Japanese patent publication H02-469    [Patent document 3] Laid-open Japanese patent publication H02-231089